Studies fronn the laboratory ofthe PI have focused on elucidating the components, mechanisms, and roles of bioactive sphingolipids, especially ceramide with its emerging roles In regulating cell growth, differentiation, senescence, and inflammation. However, the study of these pathways has been complicated by their multiplicity, metabolic interactions, sub cellular compartmentalization, and lack of molecular tools. These considerations led us to the overall hypothesis that the specific cellular functions of ceramide are dictated by the sub cellular site of ceramide generation which in turn depends on which of the enzymes of ceramide metabolism regulates its site-specific formation. In this proposal we will focus on the neutral sphingomyelinase (SMase) pathway focusing on an extended family of enzymes identified by the PI and others. Specifically, we will address the following alms: 1) To define cellular mechanisms of regulation of nSMases. We will investigate the specific hypothesis that defined signaling pathways lead to the activation/regulation of n-SMase2 in response to TNF and retinoic acid. We will also study the mechanisms by which oxidative stress regulates the activation of ma-nSMase, which we recently identified as a novel mitochondrial sphingomyelinase. 2) To determine molecular and biochemical mechanisms of activation of nSMases. We will determine the mechanisms by which nSMase2 and ma-nSMase interact with phosphatidylserine and cardiolipin, respectively, as activating lipids, and we will determine the mechanisms of phosphorylation and palmitoylation and their roles in activation and translocation of nSMases. 3) To determine the role of nSMases in generating a specific 'pool' of ceramide with specific downstream functions. We will test the specific hypothesis that nSMase2 and ma-nSMase act on a distinct pool of sphingomyelin that resides In the inner leaflet ofthe plasma membrane and in the outer leaflet ofthe mitochondrion, respectively, leading to the corresponding subset of ceramide, with its own specific functions. Taken together, these results should provide a foundation for the study of molecularly-distinct sphingolipid-mediated pathways of cell regulation. Moreover, molecular studies on nSMase2 and ma-nSMsae would also impact our understanding of the mechanisms of an emerging and novel family of phospholipid phosphodiesterases. These pathways are of direct significance to the biology of stress responses and specifically to cancer biology and inflammation.